Anti-galactofuranose antibodies for detecting and treating aspergillosis

ABSTRACT

It is provided novel anti-galactofuranose antibodies and their use for diagnosis of and/or treating aspergillosis, and for the design of chimeric antigen receptor T-cells, wherein single chain variable fragment of the antibodies, such as a heavy chain variable region or a light chain variable region, is fused via a spacer and a transmembrane domain to a signaling endodomain to generate an expression cassette that will be integrated into a T cell.

TECHNICAL FIELD

It is provided novel anti-galactofuranose antibodies and their use fordiagnostic and/or treating aspergillosis.

BACKGROUND

Members of the Aspergillus genus are opportunistic fungal pathogens thatcan cause the severe, often fatal disease, invasive aspergillosis (IA),in immunocompromised patients. A. fumigatus is the most common speciesto cause human disease, and can cause a number of severe pulmonarydiseases. The current diagnostic test for aspergillosis is anantibody-based assay that has high rates of false positives, attributedto the lack of specificity and sensitivity of the antibody being used.

The mold A. fumigatus sheds immunogenic polysaccharides from its cellwall. This fungus secretes numerous polysaccharides that can elicit animmune response. In particular, the presence of the fungalpolysaccharide, galactomannan, in bodily fluids is used as a marker ofinfection. An antibody raised against the galactofuranose portion ofgalactomannan is used in a diagnostic enzyme-linked immunosorbent assay(EIA) kit for the detection of fungal infections. This kit lacks bothspecificity and sensitivity, and routinely gives false positive results.As such, there is a need to develop more specific and sensitiveantibodies compared to the one currently used.

It is thus highly desired to be provided with new antibodies againstAspergillus fumigatus and method of using same to diagnose and treatinfections.

SUMMARY

It is provided an antibody, or a functional fragment thereof, comprisingat least one of:

-   -   a) heavy chain encoded by the DNA sequence consisting of SEQ ID        NO: 5;    -   b) a heavy chain amino acid sequence consisting of SEQ ID NO: 6;    -   c) a light chain encoded by the DNA sequence consisting of SEQ        ID NO: 7;    -   d) a light chain amino acid sequence consisting of SEQ ID NO: 8;    -   e) a sequence with at least 85%, 90%, or alternatively 95%        sequence identity to a), b) c) or d); and    -   t) a combination thereof.

It is also provided an antibody or a functional fragment thereof,comprising at least one of:

-   -   a) heavy chain encoded by the DNA sequence consisting of SEQ ID        NO: 1;    -   b) a heavy chain amino acid sequence consisting of SEQ ID NO: 2;    -   c) a light chain encoded by the DNA sequence consisting of SEQ        ID NO: 3;    -   d) a light chain amino acid sequence consisting of SEQ ID NO: 4;        and    -   e) a combination thereof.

In another embodiment, the antibody comprises a heavy chain variableregion encoded by the nucleotide sequence set forth in SEQ ID NO: 1.

In a further embodiment, the antibody comprises a heavy chain variableregion consisting of SEQ ID NO: 2.

In an additional embodiment, the antibody comprises a light chainvariable region encoded by the nucleotide sequence set forth in SEQ IDNO: 3.

In another embodiment, the antibody comprises a light chain variableregion consisting of SEQ ID NO: 4.

In another embodiment, the antibody comprises a heavy chain variableregion encoded by the nucleotide sequence set forth in SEQ ID NO: 5.

In a further embodiment, the antibody comprises a heavy chain variableregion consisting of SEQ ID NO: 6.

In an additional embodiment, the antibody comprises a light chainvariable region encoded by the nucleotide sequence set forth in SEQ IDNO: 7.

In another embodiment, the antibody comprises a light chain variableregion consisting of SEQ ID NO: 8.

It is also encompassed herein are hybridoma cell lines producing theantibodies disclosed herein.

It is further provided a method of detecting aspergillosis in a patientcomprising:

-   -   a) contacting the antibody as defined herein or a fragment        hereof with a sample of said patient; and    -   b) detecting binding of the antibody to epitopes in the sample,        wherein binding of the antibody is indicative of the presence of        an Aspergillus species in the sample.

In an embodiment, the binding of the antibody encompassed herein isdetected by an immunoassay, such as e.g. a radioimmunoassay, a Westernblot assay, an immunofluorescence assay, an enzyme immunoassay, animmunoprecipitation assay, a chemiluminescence assay, anelectrochemiluminescence assay, an immunohistochemical assay, animmunoelectrophoresis assay, a dot blot assay, or a slot blot assay.

In a particular embodiment, the antibody provided herein is used asELISA reagents. It is provided an ELISA kit comprising the antibodydisclosed herein.

In an embodiment, the Aspergillus species is Aspergillus fumigatus,Aspergillus flavus, Aspergillus niger, Aspergillus terreus, orAspergillus nidulans.

In an embodiment, the method of detecting aspergillosis as describedherein comprises also contacting an anti-galactofuranose in combinationwith the antibody described herein.

It is also provided a method of treating aspergillosis in a patientcomprising administering the antibody defined herein or a fragmentthereof to said patient.

In an embodiment, the method of treating aspergillosis as describedherein comprises also administering an anti-galactofuranose antibody incombination with the antibody described herein.

In an embodiment, it is provided an expression cassette expressing achimeric antigen receptor (CAR) comprising a heavy chain variable regionand/or a light chain variable region as described herein.

In another embodiment, it is provided a vector comprising the expressioncassette defined herein and a T cell which comprises the expressioncassette as defined herein.

It is additionally provided a method for making a T cell which comprisesthe step of introducing an expression cassette as defined herein into aT cell.

It is further provided a pharmaceutical composition which comprises a Tcell as defined herein together with a pharmaceutically acceptablecarrier, diluent or excipient.

In an embodiment, the antibody is a humanized antibody, a monoclonalantibody or a polyclonal antibody.

In a further embodiment, the antibody is a mouse antibody, a goatantibody, a human antibody or a rabbit antibody.

In another embodiment, the antibody comprises an epitope bindingfragment selected from the group consisting of: Fv, F(ab′) and F(ab′)₂.

In an additional embodiment, the antibody comprises a fluorochrome or alabeling molecule.

In an embodiment, the labeling molecule is biotin, peroxidase, alkalinephosphatase, glucoamylase, an oligonucleotide labeling, a radiolabel, ametal or β-galactosidase.

In another embodiment, the antibody binds to agalactofuranose-containing oligosaccharide motif present in aspergillusgalactomannan (GM).

In an embodiment, the antibody binds to mono-, di-, tri- andtetrasaccharides of galactofuranose.

In an embodiment, the galactofuranose oligosaccharide is:

In an embodiment, the antibody is for detecting or treatingaspergillosis.

In another embodiment, the antibody is attached to a solid support.

It is also provided a composition comprising the antibody or afunctional fragment thereof as defined herein and a carrier.

In an embodiment, the composition comprises an expression cassetteexpressing a chimeric antigen receptor (CAR) and a single chain variablefragment of the antibody.

In another embodiment, the single chain variable fragment of theantibody is a heavy chain variable region or a light chain variableregion.

In a further embodiment, the single chain variable fragment is fused viaa spacer and a transmembrane domain to a signaling endodomain.

In an embodiment, the composition comprises a T cell, the T cellexpressing the expression cassette.

In another embodiment, the composition comprises anotheranti-galactofuranose antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

Reference will now be made to the accompanying drawings.

FIG. 1 illustrates IgM monoclonal antibodies from vaccinated micerecognizing aspergillus GM. Binding of anti-Galf monoclonal antibodiesor antibodies from vaccinated mice to wild-type and GM deficient (Δugm1)A. fumigatus as detected by indirect immunofluorescence with anAlexafluor 488 anti-mouse-IgM secondary and hyphae were counterstainedwith Draq5,

FIG. 2 illustrates the recognition of anti-Galf IgM monoclonalantibodies recognizing of galactofuranose oligosaccharides showing thespecificity of BR1 (A) and BR2 (B) antibodies as measured by ELISA usingthe indicated panel of BSA-Galf glycoconjugates, and the structure ofthe minimum epitopes bound by BR1 and BR2 (C).

DETAILED DESCRIPTION

In accordance with the present description, there is provided novelanti-galactofuranose antibodies and their use for diagnostic and/ortreating aspergillosis.

The mold A. fumigatus sheds immunogenic polysaccharides from its cellwall. In particular, the presence of the fungal polysaccharide,galactomannan, in bodily fluids is used as a marker of infection.Chimeric antigen receptor T-cell (CAR T-cell) technology hasdemonstrated that reprogrammed T-cells against fungal polysaccharidesmay prove to be an effective means to augment the host immune responseduring infection. Accordingly, it is proposed that antigen recognitionsequences as provided herewith from anti-galactofuranose monoclonalantibodies can be used to construct chimeric T-cell antigen receptorsthat recognize galactofuranose within the fungal cell wall since theseepitopes are absent from humans.

A glycoconjugate vaccine approach was used to immunize mice againstneutropenic fungal infections. As a result of vaccination, the miceproduced high antibody titers against the native polysaccharide whichwere further affinity matured after challenge with A. fumigatus.Splenocytes were isolated from vaccinated mice that survived thechallenge, and monoclonal antibody producing hybridomas were generated.The minimal epitope for the antibodies produced by hybridomas producingBR1 and BR2 are a beta-(1-5)-linked galactofuranose disaccharide and agalactofuranose monosaccharide, respectively. Subsequent testing ofthese antibodies on various polysaccharide-deficient fungi demonstratedthat these antibodies are highly specific for wild-type Aspergillus.

It is thus provided two antibodies, BR1 and BR2 which are IgM antibodiesthat can bind multiple antigen epitopes simultaneously. The providedantibodies bind shorter galactofuranose oligosaccharides than theexisting monoclonal antibodies.

In an embodiment, it is provided an antibody identified as BR1comprising the heavy chain encoded by the DNA sequence consisting of:

(SEQ ID NO: 1) ATGGATTTTGGGCTGATTTTTTTTATTGTTGCTCTTTTAAAAGGGGTCCAGTGTGAGGTGAAGCTTCTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTAATCCAGATAGCAGTACGATAAACTATACGCCATCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCAAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGACCGAGAGGTTACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGAGAGTCAGTCCTTCCCAAATGTCTTCCCCCTCGTCTCCTGCGAGAGCCCCCTGTCTGATAAGAATCTGGTGGCCATGGGCTGCCTGGCCCGGGACTTCCTGCCCAGCACCATTTCCTTCACCTGGAACTACCAGAACAACACTGAAGTCATCCAGGGTATCAGAACCTTCCCAACACTGAGGACAGGGGGCAAGTACCTAGCCACCTCGCAGGTGTTGCTGTCTCCCAAGAGCATCCTTGAAGGTTCAGATGAATACCTGGTATGCAAAATCCACTACGGAGGCAAAAACAGAGATCTGCATGTGCCCATTCCAGCTGTCGCAGAGATGAACCCCAATGTAAATGTGTTCGTCCCACCACGGGATGGCTTCTCTGGCCCTGCACCACGCAAGTCTAAACTCATCTGCGAGGCCACGAACTTCACTCCAAAACCGATCACAGTATCCTGGCTAAAGGATGGGAAGCTCGTGGAATCTGGCTTCACCACAGATCCGGTGACCATCGAGAACAAAGGATCCACACCCCAAACCTACAAGGTCATAAGCACACTTACCATCTCTGAAATCGACTGGCTGAACCTGAATGTGTACACCTGCCGTGTGGATCACAGGGGTCTCACCTTCTTGAAGAACGTGTCCTCCACATGTGCTGCCAGTCCCTCCACAGACATCCTAACCTTCACCATCCCCCCCTCCTTTGCCGACATCTTCCTCAGCAAGTCCGCTAACCTGACCTGTCTGGTCTCAAACCTGGCAACCTATGAAACCCTGAATATCTCCTGGGCTTCTCAAAGTGGTGAACCACTGGAAACCAAAATTAAAATCATGGAAAGCCATCCCAATGGCACCTTCAGTGCTAAGGGTGTGGCTAGTGTTTGTGTGGAAGACTGGAATAACAGGAAGGAATTTGTGTGTACTGTGACTCACAGGGATCTGCCTTCACCACAGAAGAAATTCATCTCAAAACCCAATGAGGTGCACAAACATCCACCTGCTGTGTACCTGCTGCCACCAGCTCGTGAGCAACTGAACCTGAGGGAGTCAGCCACAGTCACCTGCCTGGTGAAGGGCTTCTCTCCTGCAGACATCAGTGTGCAGTGGCTTCAGAGAGGGCAACTCTTGCCCCAAGAGAAGTATGTGACCAGTGCCCCGATGCCAGAGCCTGGGGCCCCAGGCTTCTACTTTACCCACAGCATCCTGACTGTGACAGAGGAGGAATGGAACTCCGGAGAGACCTATACCTGTGTTGTAGGCCACGAGGCCCTGCCACACCTGGTGACCGAGAGGACCGTGGACAAGTCCACTGGTAAACCCACACTGTACAATGTCTCCCTGATCATGTCTGACACAGGCGGCACCTGCTATTGA.

In an embodiment, said BR1 antibody comprises the following heavy chainamino acid sequence:

(SEQ ID NO: 2) MDFGLIFFIVALLKGVQCEVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCARPRGYYAMDYWGQGTSVTVSSESQSFPNVFPLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIQGIRTFPTLRTGGKYLATSQVLLSPKSILEGSDEYLVCKIHYGGKNRDLHVPIPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMSDTGGTCY.

In an embodiment, it is provided an antibody identified as BR1comprising the light chain encoded by the DNA sequence consisting of:

(SEQ ID NO: 3) ATGGAGTCACAGATTCAGGTCTTTGTATTCGTGTTTCTCTGGTTGTCTGGTGTTGACGGAGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCGGCATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGT GTTAG.

In an embodiment, said BR1 antibody comprises the following light chainamino acid sequence:

(SEQ ID NO: 4) MESQIQVFVFVFLWLSGVDGDIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

in an embodiment, it is provided an antibody identified as BR2comprising the heavy chain encoded by the DNA sequence consisting of:

(SEQ ID NO: 5) ATGGATTTTGGGCTGATTTTTTTTATTGTTGCTCTTTTAAAAGGGGTCCAGTGTGAGGTGAAGCTTCTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGAAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAATTGGGTCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAGTTAATCCAGATAGCAGTACGATAAACTATACGCCTTCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTATCTGCAAATGAGTAAAGTGATATCTGAGGACACAGCCCTTTATTTCTGTGCAAGACCGAGGGGTAACTACGGTATAGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGAGAGTCAGTCCTTCCCAAATGTCTTCCCCCTCGTCTCCTGCGAGAGCCCCCTGTCTGATAAGAATCTGGTGGCCATGGGCTGCCTGGCCCGGGACTTCCTGCCCAGCACCATTTCCTTCACCTGGAACTACCAGAACAACACTGAAGTCATCCAGGGTATCAGAACCTTCCCAACACTGAGGACAGGGGGCAAGTACCTAGCCACCTCGCAGGTGTTGCTGTCTCCCAAGAGCATCCTTGAAGGTTCAGATGAATACCTGGTATGCAAAATCCACTACGGAGGCAAAAACAGAGATCTGCATGTGCCCATTCCAGCTGTCGCAGAGATGAACCCCAATGTAAATGTGTTCGTCCCACCACGGGATGGCTTCTCTGGCCCTGCACCACGCAAGTCTAAACTCATCTGCGAGGCCACGAACTTCACTCCAAAACCGATCACAGTATCCTGGCTAAAGGATGGGAAGCTCGTGGAATCTGGCTTCACCACAGATCCGGTGACCATCGAGAACAAAGGATCCACACCCCAAACCTACAAGGTCATAAGCACACTTACCATCTCTGAAATCGACTGGCTGAACCTGAATGTGTACACCTGCCGTGTGGATCACAGGGGTCTCACCTTCTTGAAGAACGTGTCCTCCACATGTGCTGCCAGTCCCTCCACAGACATCCTAACCTTCACCATCCCCCCCTCCTTTGCCGACATCTTCCTCAGCAAGTCCGCTAACCTGACCTGTCTGGTCTCAAACCTGGCAACCTATGAAACCCTGAATATCTCCTGGGCTTCTCAAAGTGGTGAACCACTGGAAACCAAAATTAAAATCATGGAAAGTCATCCCAATGGCACCTTCAGTGCTAAGGGTGTGGCTAGTGTTTGTGTGGAAGACTGGAATAACAGGAAGGAATTTGTGTGTACTGTGACTCACAGGGATCTGCCTTCACCACAGAAGAAATTCATCTCAAAACCCAATGAGGTGCACAAACATCCACCTGCTGTGTACCTGCTGCCACCAGCTCGTGAGCAACTGAACCTGAGGGAGTCAGCCACAGTCACCTGCCTGGTGAAGGGCTTCTCTCCTGCAGACATCAGTGTGCAGTGGCTTCAGAGAGGGCAACTCTTGCCCCAAGAGAAGTATGTGACCAGTGCCCCGATGCCAGAGCCTGGGGCCCCAGGCTTCTACTTTACCCACAGCATCCTGACTGTGACAGAGGAGGAATGGAACTCCGGAGAGACCTATACCTGTGTTGTAGGCCACGAGGCCCTGCCACACCTGGTGACCGAGAGGACCGTGGACAAGTCCACTGGTAAACCCACACTGTACAATGTCTCCCTGATCATGTCTGACACAGGCGGCACCTGCTATTGA.

In an embodiment, said BR2 antibody comprises the following heavy chainamino acid sequence:

(SEQ ID NO: 6) MDFGLIFFIVALLKGVQCEVKLLESGGGLVQPGGSLKLSCEASGFDFSRYWMNWVRQAPGKGLEWIGEVNPDSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVISEDTALYFCARPRGNYGIDYWGQGTSVTVSSESQSFPNVFPLVSCESPLSDKNLVAMGCLARDFLPSTISFTWNYQNNTEVIQGIRTFPTLRTGGKYLATSQVLLSPKSILEGSDEYLVCKIHYGGKNRDLHVPIPAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGKPTLYNVSLIMSDTGGTCY.

In an embodiment, it is provided an antibody identified as BR2comprising the light chain encoded by the DNA sequence consisting of:

(SEQ ID NO: 7) ATGGAGTCACAGATTCAGGTCTTTGTATTCGTGTTTCTCTGGTTGTCTGGCGTTGACGGAGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTGGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAATATATAATTACTTCTGTTGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACCACTGATTTACTCGTCATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCATCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAACAACATTTTAGTATTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGT GTTAG.

In an embodiment, said BR2 antibody comprises the following light chainamino acid sequence:

(SEQ ID NO: 8) MESQIQVFVFVFLWLSGVDGDIVMTQSHKFMSTSVGDRVSITCKASQYIITSVAWYQQKPGQSPKPLIYSSSYRYTGVPDRFTGSGSGTDFTFTIISVQAEDLAVYYCQQHFSIPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

In an embodiment, the antibody encompassed herein is a humanizedantibody, a monoclonal antibody or a polyclonal antibody.

Accordingly it is disclosed humanized antibodies and antibodies whoseprotein sequences can be modified to increase their similarity toantibody variants produced naturally in humans. Humanization can benecessary when the process of developing a specific antibody involvesgeneration in a non-human immune system (such as that in mice). Antibodyhumanization methods are well known and designed to produce a moleculewith minimal immunogenicity when applied to humans, while retaining thespecificity and affinity of the parental non-human antibody. The proteinsequences of antibodies produced in this way are partially distinct fromhomologous antibodies occurring naturally in humans, and are thereforepotentially immunogenic when administered to human patients.

Humanized antibodies encompassed herein can be produced via enrichmenttechnologies such as phage display or immunization of transgenic micebearing the antibody human gene repertoire have provided powerful meansto generate human antibodies.

In another embodiment, the antibody is a mouse antibody, a goatantibody, a human antibody or a rabbit antibody.

In an embodiment, the antibody comprises an epitope binding fragmentselected from the group consisting of: Fv, F(ab′) and F(ab′)₂.

In another embodiment, the antibody comprises a heavy chain variableregion encoded by the nucleotide sequence set forth in SEQ ID NO: 1.

In a further embodiment, the antibody comprises a heavy chain variableregion consisting of SEQ ID NO: 2.

In an additional embodiment, the antibody comprises a light chainvariable region encoded by the nucleotide sequence set forth in SEQ IDNO: 3.

In another embodiment, the antibody comprises a light chain variableregion consisting of SEQ ID NO: 4.

In another embodiment, the antibody comprises a heavy chain variableregion encoded by the nucleotide sequence set forth in SEQ ID NO: 5.

In a further embodiment, the antibody comprises a heavy chain variableregion consisting of SEQ ID NO: 6.

In an additional embodiment, the antibody comprises a light chainvariable region encoded by the nucleotide sequence set forth in SEQ IDNO: 7.

In another embodiment, the antibody comprises a light chain variableregion consisting of SEQ ID NO: 8.

It is also provided a composition comprising the antibody as describedherein and a carrier. The antibodies described herein may be employed inadmixture with a suitable physiological or pharmaceutical carrier. Suchcompositions comprise a therapeutically effective amount of theantibody, and a physiologically or a pharmaceutically acceptable carrieror excipient. Such a carrier includes but is not limited to saline,buffered saline, dextrose, water, glycerol, ethanol, and combinationsthereof. The formulation should suit the mode of administration.

An antibody as defined herein, can be administered alone or incombination with other antibodies directed toward other complementarytargets.

In an embodiment, the antibodies provided herein can be used in adetection kit for aspergillosis.

In an embodiment, the antibodies provided herein further comprises afluorochrome or a labeling, such as for example, biotin, peroxidase, anoligonucleotide labeling, a radiolabel, a metal, alkaline phosphatase,glucoamylase or 3-galactosidase.

In another embodiment, the level of A. fumigatus are measured byimmunoassay using the antibodies provided herein.

In an embodiment, the immunoassay is a radioimmunoassay, a Western blotassay, an immunofluorescence assay, an enzyme immunoassay, animmunoprecipitation assay, a chemiluminescence assay, anelectrochemiluminescence assay, an immunohistochemical assay, animmunoelectrophoresis assay, a dot blot assay, a slot blot assay, anImmunoPET/MR, or immunoPCR assay. The antibodies provided herein can befor example can be attached on a solid support.

In a particular embodiment, the antibodies provided herein are used asELISA reagents. Encompassed herein is an ELISA kit comprising theantibodies disclosed herein.

The antibodies are collectively assembled in a kit with conventionalimmunoassay reagents for detection of A. fumigatus using an immunoassay.The kit may optionally contain both monoclonal and polyclonal antibodiesand a standard for determining the presence of A. fumigatus in a sample.The kit containing these reagents provides for simple, rapid, on sitedetection. The antibodies described above are used as the basic reagentsof a number of different immunoassays to determine the presence of A.fumigatus in a sample. The antibodies are employed in any type ofimmunoassay, whether qualitative or quantitative.

In another embodiment, the antibodies described herein further comprisea detectable label which is for example an enzyme. In more preferredembodiments, the enzyme is alkaline phosphatase, peroxidase, orβ-galactosidase. In another embodiment, the enzyme produces a solublereaction product.

In another embodiment, the enzyme produces a soluble or an insolublereaction product. In another embodiment, the kit further comprises asubstrate for the enzyme. Such immunoassays are also referred toenzyme-linked immunosorbent assays (ELISA).

The antibodies described above can also be employed in a lateral flowassay and/or an immunostrip. A solid phase format such as an immunostripconsist of test strips comprising multiple porous components, membranesand filters, through which liquid sample is drawn by capillary action.

One or more of the antibodies described above are employed in anyheterogeneous or homogeneous, sandwich or competitive immunoassay forthe detection of aspergillosis. Either the antibodies provided hereinare labelled with a detectable label or coupled to a solid phase.Methods for coupling antibodies to solid phases are well known to thoseskilled in the art (e.g, direct coupling to a surface or also includesprotein NG catching of the antibody). In accordance with the immunoassaymethod, the sample containing A. fumigatus is reacted with the antibodyfor a sufficient amount of time under conditions that promote thebinding of antibody to its antigen protein in the sample. A physicalmeans is employed to separate reagents bound to the solid phase fromunbound reagents such as filtration of particles, cell/particle sortingby FACS, decantation of reaction solutions from coated tubes or wells,magnetic separation, capillary action, and other means known to thoseskilled in the art. It will also be understood that a separate washingof the solid phase may be included in the method.

It is also encompassed herein are hybridoma cell lines producing theantibodies disclosed herein.

In an embodiment, it is encompassed the use of specific recognitionsequences for the design of chimeric antigen receptor T-cells. Chimericantigen receptors are known to be proteins which, in their usual format,combine the specificity of a monoclonal antibody (mAb) with the effectorfunction of a T-cell. Their usual form is that of a type I transmembranedomain protein with an antigen recognizing amino terminus, a spacer, atransmembrane domain all connected to a compound endodomain whichtransmits T-cell survival and activation signals. For example, a commonform of these molecules consist of fusions of single-chain variablefragments (scFv) derived from monoclonal antibodies such as theantibodies encompassed herein which recognize a target antigen, fusedvia a spacer and a transmembrane domain to a signaling endodomain. Suchmolecules result in activation of the T-cell in response to recognitionby the scFv of its target. When T cells express such a CAR, theyrecognize and kill target cells that express the target antigen. It isthus encompass CARs developed against A. fumigatus associated antigensusing the antibody sequences and fragments provided herein.

In an embodiment, it is encompassed an expression cassette expressing achimeric antigen receptor (CAR) comprising a heavy chain variable regionand/or a light chain variable region as described herein. It is thusencompassed a vector comprising the expression cassette defined hereinand a T cell which comprises the expression cassette as defined herein.

Also encompassed is a method for making a T cell or NK cell whichcomprises the step of introducing an expression cassette as definedherein into a T cell or NK cell. Also encompassed is a pharmaceuticalcomposition which comprises a T cell as defined herein together with apharmaceutically acceptable carrier, diluent or excipient.

It is further encompassed combining BR1 and/or BR2 with anotheranti-galactofuranose antibody. It is provided that such combination mayexhibit increased sensitivity as compared to combination of knownantibodies, including existing commercialized anti-galactofuranoseantibody, such as for example but not limited to EB-A2 or AP3.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

Example I Isolation and Characterization of BR1 and BR2

Synthesis of TT-Galf₄

Tetanus toxoid (Statens Serum Institute) in PBS was added to propargyllinker to react overnight. Propargylated protein was then concentratedusing a gel filtration column. BCA protein assay (Thermo Fisher) andMALIDI-TOF MS were used to quantify the amount of protein and determineamino group modifications, respectively. Next, the propargylated proteinwas conjugated to Galf₄-N₃ by Azide-Alkyne Huisgen Cycloaddition aspreviously described. Briefly, the propargylated protein was incubatedovernight in a sealed glass vial with the oligosaccharide in thepresence of copper powder. After incubation, the reaction was quenchedwith EDTA and the protein washed with PBS. Finally, the conjugate waspurified by gel filtration column. The resulting protein concentrationand degree of oligosaccharide incorporation were assessed by BCA proteinassay (Thermo Fisher) and MALDI-TOF MS respectively.

Immunizations

The day prior to immunization, TT-Galf₄ and alum (Alhydrogel®, BrenntagAG) were diluted to working concentrations and incubated on a rotaryshaker overnight. Animals were immunized on day 0, 14, and 21 byadministering a total of 6 μg TT-Galf₄ and 30 μg alum intraperitoneally,and 4 μg TT-Galf₄ and 20 μg alum subcutaneously. On day 31 serum wascollected either from submandibular puncture (non-lethal) or cardiacpuncture (lethal). Mice were infected on day 31 as detailed below.

Anti-Galf₄ ELISA and Monoclonal Antibody Epitope Mapping

High binding ELISA plates were coated overnight at 4° C. with 0.1 μg ofbovine serum albumen (BSA)-Galf₄ (or other BSA glycoconjugates used forepitope mapping) in 100 μL PBS per well. Wells were washed withphosphate buffered saline+0.05% Tween™-20(PBS-T) and blocked with 1% BSAprepared in PBS. After washing, serially diluted serum samples ormonoclonal antibodies in 0.1% BSA in PBS-T were added to the wells andincubated for 2 hours. Following washing, plates were incubated with1:5000 diluted anti-mouse IgG or anti-mouse IgM secondary antibodiesconjugated to horseradish peroxidase (Jackson ImmunoResearchLaboratories Inc.). After washing, the wells were developed with 200 μLtetramethylbenzidine (TMB) and the reaction was stopped with 100 μL 2Nsulphuric acid. The absorbance was then read at 450 nm.

Immunofluorescence

Poly-D-lysine coverslips in 24-well plates were inoculated with 1×10⁵conidia in DMEM media and grown for 10 hours at 37° C. in 5% CO₂. Hyphaewere gently washed three times between each step with PBS except wherenoted. Fungi were fixed with 4% PFA and blocked with a 50% fetal bovineserum, 1.5% BSA solution prepared in PBS. Serum samples were thendiluted 1:500 in blocking solution and were added to unwashed coverslipsfor 1 hour at room temperature. Following washing, hyphae were incubatedfor an additional hour at room temperature in the dark in eitheranti-mouse or anti-rabbit IgG, A, M secondary antibodies conjugated toAlex Fluor® 488 (Invitrogen) that were diluted 1:500 in blocking buffer.Hyphae were then counterstained with DRAQ™5 (Invitrogen) diluted 1:1000in PBS, washed and fixed in PFA. After a last set of washes, coverslipswere mounted on slides and imaged using an LSM 780.

Infection Models

For systemic infection models, mice were infected with 3×10⁷conidia/mouse intravenously by tail vein injection on day 31 afterinitiation of immunization. For studies of pulmonary infection, micewere rendered neutropenic by treatment with 200 μg of anti-Ly6G (clone1A8, Bio X Cell) antibodies intraperitoneally every 48 hours beginningthe day prior to infection. On the day of infection, mice wereanaesthetized by isoflurane then infected endotracheally with 1×10⁷conidia of A. fumigatus in a 504 volume of PBS containing 0.1% tween-80.

Generation of Monoclonal Antibodies

Monoclonal antibody producing hybridomas were generated from immunizedmice surviving A. fumigatus challenge. Mice were immunized andchallenged as described above. Fourteen days following infection, thespleens of mice that survived infection were harvested and transportedfresh to MédiMabs (Montreal, Qc) for commercial immortalization of thesplenocytes. Hybridomas were screened for the production of anti-Galf₄antibodies by BSA-Galf₄ ELISA, The eleven stable hybridomas that werereactive by BSA-Galf₄ were then tested for their reactivity with nativeGM by immunofluorescence using hyphae of the wild-type (Af293) andGalf-deficient A. fumigatus (Δugm1).

To determine if anti-Galf₄ antibodies could mediate protection againstA. fumigatus challenge, monoclonal anti-Galf₄ antibodies were generated.Hybridomas were generated from splenocytes obtained from immunized micethat survived an A. fumigatus challenge. From a total of 600 hybridomas,11 clones produced antibodies that bound to BSA-Galf₄ by ELISA. Of the11 clones, two IgM antibody producing hybridomas (BR1 and BR2) wereidentified that produced antibodies specific against native GMpolysaccharide as determined by immunofluorescence (FIG. 1). Theantigen-specificity of the antibodies produced by these two clones wasfurther assessed by testing antibody reactivity to a panel ofglycoconjugates. Using this technique, the smallest recognizable epitoperecognized by BR1 was determined to be a disaccharide of β(1-5) linkedGalf (se FIG. 2C) while BR2 was able to recognize Galf monosaccharide(FIG. 2D). To determine the ability of these antibodies to mediateprotection against infection, naïve neutropenic mice were administeredBR1 or BR2 antibodies beginning on the day of infection and 48 hoursafter. No difference in survival was observed between naïve mice, orthose receiving either BR1 or BR2 antibody therapy.

While the present description has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications and this application is intended to cover anyvariations, uses, or adaptations including such departures from thepresent disclosure as come within known or customary practice within theart and as may be applied to the essential features hereinbefore setforth, and as follows in the scope of the appended claims.

What is claimed is:
 1. An antibody or a functional fragment thereof,comprising at least one of: a) a heavy chain encoded by the DNA sequenceconsisting of SEQ ID NO: 5; b) a heavy chain amino acid sequenceconsisting of SEQ ID NO: 6; c) a light chain encoded by the DNA sequenceconsisting of SEQ ID NO: 7; d) a light chain amino acid sequenceconsisting of SEQ ID NO: 8; and e) a combination thereof.
 2. Theantibody or a functional fragment thereof of claim 1, wherein saidantibody is a humanized antibody, a monoclonal antibody or a polyclonalantibody.
 3. The antibody or a functional fragment thereof of claim 1,wherein said antibody is a mouse antibody, a goat antibody, a humanantibody or a rabbit antibody.
 4. The antibody or a functional fragmentthereof of claim 1, wherein said antibody comprises an epitope bindingfragment selected from the group consisting of: Fv, F(ab′) and F(ab′)₂.5. The antibody or a functional fragment thereof of claim 1, whereinsaid antibody comprises a fluorochrome or a labeling molecule.
 6. Theantibody or a functional fragment thereof of claim 5, wherein saidlabeling molecule is biotin, peroxidase, alkaline phosphatase, anoligonucleotide labeling, a radiolabel, a metal, glucoamylase orB-galactosidase.
 7. The antibody or a functional fragment thereof ofclaim 1, wherein said antibody comprises the heavy chain encoded by theDNA sequence consisting of SEQ ID NO: 5; the heavy chain amino acidsequence consisting of SEQ ID NO: 6; the light chain encoded by the DNAsequence consisting of SEQ ID NO: 7; and the light chain amino acidsequence consisting of SEQ ID NO: 8, binds to a galactofuranoseoligosaccharide.
 8. The antibody or a functional fragment thereof ofclaim 7, wherein said antibody detects a polysaccharide comprising amono-, di-, tri-, or tetrasaccharides of galactofuranose.
 9. Theantibody or a functional fragment thereof of claim 7, wherein saidgalactofuranose oligosaccharide is:


10. The antibody or a functional fragment thereof of claim 1, saidantibody is attached to a solid support.
 11. A composition comprisingthe antibody or a functional fragment thereof of claim 1 and a carrier.12. The antibody or a functional fragment thereof of claim 9, whereinX=0 to
 20. 13. The antibody or a functional fragment thereof of claim 9,wherein X=0 to
 10. 14. The antibody or a functional fragment thereof ofclaim 9, wherein X=0 to 3.